(1) Field of the Invention
This invention relates to a serum matrix or serum-derived composition containing lipids, such as a lipid calibrator, which has been stabilized against inhomogeneity which results from lyophilization and reconstitution with water.
(2) State of the Prior Art
Automated clinical analyzers such as those which rely upon radiometric detection of a change in absorption require calibration using a "standard" calibrator. Such calibrators permit two or more known concentrations of an analyte of choice to be fixed at particular levels, e.g., color densities, as read by the analyzer, and from these a curve is derived for use with readings of unknown analyte concentrations.
For example, lipid calibrators are used for calibrating the analysis of cholesterol and triglycerides. Such calibrators, as with other calibrators, are either prepared fresh on a daily basis, or as is more common, are stored in a lyophilized condition for reconstitution at a later date. It is well-known that lipoproteins will not readily tolerate lyophilization, but rather produce an inhomogeneous, turbid reconstituted solution, due to instability of particular lipoproteins to the lyophilization process. Serum and blood plasma containing lipoproteins and glucose in naturally occurring amounts have also demonstrated unacceptable turbidity after lyophilization and reconstitution.
U.S. Pat. Nos. 3,260,648 issued on July 12, 1966 and 3,955,925 issued on May 11, 1976 are illustrative of the problem. As explained in these patents, turbidity interferes with the analysis and therefore with the calibration of the analyzer. Although turbidity might be overcome by many-fold dilutions, such dilutions are time-consuming and therefore undesirable.
U.S. Pat. Nos. 3,955,925 and 4,011,045; and Japanese Patent O.P.I. No. 144724/76 are of interest, because they disclose that turbidity upon lyophilization and reconstitution can be avoided, by removing, such as by salting out, most of the lipoproteins which are the primary cause of the turbidity problem. Thereafter, desired proteins such as .alpha.-fetoprotein, which is not a lipoprotein, or glycerides of low molecular weight fatty acids, are added back. In the Japanese process, lactose is described as a useful additive for the lyophilization step, presumably for the bulking effect described hereinafter. The technique described in U.S. Pat. No. 4,011,045 however is seemingly of limited utility, being disclosed as a method for preparing a useful standard only for triglycerides.
Because fresh, daily preparation of the calibrator is usually impractical as an alternative to lyophilization and reconstitution, what is needed is an additive that will permit the reconstitution of lyophilized lipid calibrators without a significant loss of optical clarity.
Sugar and sugar derivatives have been used as bulking agents and stabilizers for reconstituted, lyophilized assay mixtures, as shown for example in U.S. Pat. No. 3,413,198. However, the assay mixtures described in this patent are not disclosed specifically as containing lipids such as cholesterol or triglyceride, and no recognition is given to an improvement in clarity to be achieved by the use of such "bulking agents."
Lactose has been used as a bulking agent for solutions in general to be lyophilized, but only where the solutions require such bulk for ease in the lyophilization process. However, the amounts of lactose used for this effect generally have been less than that found to be effective by this invention to provide clarity. Moreover, lipoproteins due to their large molecular size do not require such additional bulking agents.
Glucose is of course a sugar that is both naturally present in serum or has been added to calibrators of various kinds. However, the amount present either naturally or by addition has been ineffective to produce any clarity stabilization, as is evident from the fact that human serum, upon lyophilization, typically exhibits a drastic increase in turbidity.
Patents which relate generally to the background of lipids or other calibrators include U.S. Pat. Nos. 3,274,062; issued on Sept. 20, 1966; 3,751,381 issued Aug. 7, 1973; 3,897,363 issued July 29, 1975; and 4,007,008 issued Feb. 8, 1977.